High-throughput nucleotide sequence analysis of diverse bacterial communities in leachates of decomposing pig carcasses

High-throughput nucleotide sequence analysis of diverse bacterial

communities in leachates of decomposing pig carcasses

Seung Hak Yang

, Joung Soo Lim

, Modabber Ahmed Khan

, Bong Soo Kim

, Dong Yoon Choi

,

Eun Young Lee

and Hee Kwon Ahn

1

_{Animal Nutrition Physiology Team, National Institute of Animal Science, Jeonbuk, South Korea.}

2

_{Animal Environment Division, National Institute of Animal Science, Jeonbuk, South Korea.}

3

_{Department of Life Sciences, Hallym University, Chuncheon, Gangwon-do, South Korea.}

4

Department of Environmental and Energy Engineering, Suwon University, South Korea.

Department of Animal Biosystems Science, Chungnam National University, Daejeon, South Korea.

Abstract

The leachate generated by the decomposition of animal carcass has been implicated as an environmental contami-nant surrounding the burial site. High-throughput nucleotide sequencing was conducted to investigate the bacterial communities in leachates from the decomposition of pig carcasses. We acquired 51,230 reads from six different samples (1, 2, 3, 4, 6 and 14 week-old carcasses) and found that sequences representing the phylum Firmicutes pre-dominated. The diversity of bacterial 16S rRNA gene sequences in the leachate was the highest at 6 weeks, in con-trast to those at 2 and 14 weeks. The relative abundance of Firmicutes was reduced, while the proportion of Bacteroidetes and Proteobacteria increased from 3-6 weeks. The representation of phyla was restored after 14 weeks. However, the community structures between the samples taken at 1-2 and 14 weeks differed at the bacterial classification level. The trend in pH was similar to the changes seen in bacterial communities, indicating that the pH of the leachate could be related to the shift in the microbial community. The results indicate that the composition of bacterial communities in leachates of decomposing pig carcasses shifted continuously during the study period and might be influenced by the burial site.

Keywords: pig, decomposition, leachate, bacterial community, pyrosequencing.

Received: September 14, 2014; Accepted: February 10, 2015.

Introduction

Foot and mouth disease (FMD) is highly contagious and is caused by members of the genusAphthovirus, repre-sented by various serotypes, such as A, O, C, SAT-1, SAT-2, SAT-3, and Asia-1 (Brooksby, 1982; Seellers, 1995).

FMD is especially fatal in young animals as a result of myocardial damage, while the mortality rate of FMD might be low in adult animals (Sharma and Das, 1984; Woodbury, 1995; Domingo et al., 1990; Gruman and Baxt, 2004). Therefore, FMD-infected animals are slaughtered immedi-ately and buried according to guidance on animal carcass mass burial procedures developed by each country. Despite an extensive nationwide quarantine effort, South Korea ex-perienced the worst FMD epidemic in the country’s history during 2010-2011, resulting in the culling of more than 3.5 million heads ofArtiodactyla, including cattle, goat and pigs (Ahn, 2012).

Although burial is the most common method used to dispose of large numbers of deceased livestock, the poten-tial pathogenic content of the carcass associated health problems to residents living near the burial site have re-sulted in widespread public health concerns (NRC, 2002). Moreover, leachates derived from the decomposition of an-imal carcasses consist of organic intermediates, volatile chemicals, and saprotrophic organisms, which may pose a high contamination risk to soil and groundwater when leak-ing through the basement layer (Christensenet al., 1994; Rölinget al., 2001; Tianet al., 2005).

The rate at which animal carcasses decompose in the burial pit can be affected by temperature, humidity, the depth of the pit, chemical composition, and other environ-mental factors (Mannet al., 1990; McDaniel, 1991; Vasset al., 1992; Paul and Clark, 1996; Gill-King, 1997; Carter and Tibbett, 2006; Prattet al., 2010). Generally, tempera-ture and pH can accelerate microbial decomposition by changing the soil microhabitat (Paul and Clark, 1996; Blagodatskaya and Anderson, 1998). For example, the met-abolic activity of a mesophilic microbial population

Send correspondence to Seung Hak Yang. Animal Nutrition Physi-ology Team, National Institute of Animal Science, 565-851 Jeonbuk, South Korea. E-mail: y64h@korea.kr.

increased approximately two-fold with an increase in tem-perature from 10 °C to 30-35 °C (Van’tHoff, 1898; Conant

et al., 2004). Moreover, pH has a pivotal role in the type of microorganisms that predominate in different soils (Lynch and Hobbie, 1988; Matthieset al., 1997). Other environ-mental factors, including moisture, dissolved oxygen, con-ductivity and the like also play an important role in the microbial community composition (Keener et al., 2000; Pettersson M, (2004, Doctoral thesis, Lund University, Sweden). Soil properties may also affect the microbial community (Carteret al., 2007) and, hence, the carcass de-composition process (Pfeifferet al., 1998).

In a forensic study, the bodies of obese people decom-posed faster than those of average weight individuals due to the greater amount of liquid present in the tissues, which contributes to the spread and proliferation of bacteria (Campobassoet al., 2001). The high organic load of the leachate could cause drastic changes in aquifer geochemis-try and microbiology downstream of landfills (Roling et al., 2001), and leachates originating from the decomposi-tion of buried pig carcasses, as investigated here, could cause similar changes in soil and in the geochemical char-acteristics of water as well.

The methods for burying dead livestock or the charac-teristics of uncultured microbial communities in the burial site have not been studied in detail. The presence of many uncultured bacteria has been confirmed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and phospholipid fatty acid (PLFA) profil-ing. However, such studies primarily focused on specific microbial communities in landfill leachate plumes (Lud-vigsenet al., 1997; Sasakiet al., 2009; Yanget al., 2012). The advent of next generation sequencing techniques, such as bar-coded pyrosequencing, can elucidate microbial com-munity structures at a much higher resolution than previ-ously possible. Moreover, the large dataset produced by pyrosequencing enables the detection of rare microbes and to perform phylogenetic comparisons of microbes living in a particular environment (Liuet al., 2007, 2008; Hamadyet al., 2010; Kirchmanet al., 2010). Therefore, the goal of the present study was to take advantage of pyrosequencing to investigate the identities of members of microbial commu-nities in leachates originating from the decomposition of pig carcasses over a period of 98 days.

Materials and Methods

Testing device

A lab-scale system was designed based on animal car-cass mass burial procedures developed by the Korean Min-istry for Food, Agriculture, Forestry and Fisheries. The lab-scale system, (1/7 normal size), was 0.54 m (l) x 1.4 m (w) x 0.85 m (h), approximately 0.67 m3in volume, and is described in detail by Yanget al.(2012). Three lab-scale systems, each of which can accommodate an

approxi-mately 115 kg single pig carcass, were placed in a room at 35 °C. The leachate samples were collected using a peristal-tic pump from each of three containers once each week for the first month and then at 6 and 14 weeks. Serial collected sample sets from the triplicate system during decomposi-tion were used for molecular analysis. The pH was mea-sured immediately after collection using a bench-top pH meter (Orion 4 STAR, Thermo Scientific Inc., Beverly, USA), and then the leachate samples were stored at -80 °C.

PCR amplification and pyrosequencing

Total genomic DNA was extracted from leachates us-ing a Fast DNA Spin kit for soil (MP Bio, Texas, USA). Fragments of the V1-V3 regions of the 16S rDNA were am-plified using fusion primers from genomic DNA extracted from leachate samples (Huret al., 2011). Each PCR reac-tion was performed in a final volume of 50mL with 5mL of

10X Taq buffer, dNTP mixture (Takara, Shiga, Japan), 10mM of each primer and 2 U ofTaqpolymerase (Ex Taq,

Takara) using a C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, USA). Amplification reactions were per-formed as follows: 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension step at 72 °C for 7 min. After visualizing the PCR product using gel electrophoresis and the Gel Doc system (Bio-Rad), the amplicons were purified using a QIAquick PCR purifica-tion kit (Qiagen, Valencia, CA, USA). Purified amplicons from different samples (same concentration of each sam-ple) were pooled and purified using an AMPure bead kit (Agencourt Bioscience, Beverly, MA, USA). Pooled prod-ucts were amplified on the sequencing beads using emul-sion PCR, and the beads were deposited on a 454 Picotiter Plate and sequenced using a GS Junior system (Roche, Brandford, CT, USA) according to the manufacturer’s in-structions.

Analysis of pyrosequencing

distances between communities were estimated using Fast UniFrac (Hamadyet al., 2010) and visualized with princi-pal coordinate analysis (PCoA). The significances of the differences between community structures were calculated using Libshuff analysis (Singleton et al., 2001). Pyro-sequences obtained in this study were uploaded to the EMBL SRA database under the study accession number ERP002343

(http://www.ebi.ac.uk/ena/data/view/ERP002343).

Results and Discussion

The analyses of bacterial communities in leachates from different stages of the decomposition of pig carcasses were conducted by pyrosequencing of 16S rRNA gene amplicons. A total of 51,230 reads were obtained from six different sampling times, and normalized read numbers in each sample were used to compare estimated values among samples (Table 1). The minimum value of Good’s coverage was 81% (week 6 sample), and the maximum value was 90% (week 2 sample). The highest and lowest numbers of operational taxonomic units (OTUs) was observed at week

6 (1,002 OTUs) and 2 (549), respectively. The diversity of community composition changed during the periods of de-composition. The Shannon index decreased from 1 (4.10) to week 4 (3.22), increased at week 6 (4.58), and decreased again at week 14 (3.06). These results indicate that the bac-terial communities in leachates at 1 and 6 weeks consisted of various bacterial species and were complex. The pres-ence of dominant bacterial species decreased the diversity of the bacterial community observed during the early stage.

The rarefaction curves of the samples are presented in Figure 1. The highest richness of the bacterial community was detected in the 6-week sample and the lowest at 2 and 14 weeks. The dominant phylum was Firmicutes in all sam-ples, and the abundance of Proteobacteria and Bacteroi-detes was increased in the 6-week sample (Figure 2). The shift in the composition of the bacterial community was in-vestigated in more detail at the genus level. The abundance of unclassified bacteria withinTissierellagenus increased from weeks 2 to 4 (27% of total reads at week 1, > 46.1% from weeks 2 to 4). Peptostreptococcus increased at 14 weeks (38.7% of total reads). RuminalPeptostreptococcus

Table 1- Comparison of pH to the abundance of bacterial 16S rDNA sequences of leachates from decomposing pig carcasses.

Sample (week)

pH Analyzed reads Normalized reads

Average length (bp)

Observed OTUs

Estimated OTUs (Chao1)

Shannon Index Good’s coverage

1 6.59 4,100 4,100 454.44 667 2,910.31 4.10 0.87

2 6.47 5,230 4,100 457.63 549 1,977.74 3.05 0.90

3 6.46 17,035 4,100 455.34 799 5,147.62 3.45 0.84

4 6.51 7,805 4,100 454.88 650 3,177.83 3.22 0.87

6 8.79 5,674 4,100 458.89 1,002 4,349.40 4.58 0.81

14 6.48 11,386 4,100 455.65 593 2,645.86 3.06 0.88

plays an important role in the degradation of valine, iso-leucine, and leucine (Chen and Russell, 1989). The relative abundance ofAnaerosalibacterwas the highest at week 1 (over 11% of total reads); this genus was isolated from oil sludge and is reported to be a reducing species during the decomposition process (Rezguiet al., 2012).

Tissierella andTepidimicrobium are anaerobic spe-cies that reduce protein and amino acids, and these bacteria generally increased during decomposition (Harmset al., 1998; Slobodkinet al., 2006). Our present data reveal that there was a gradual reduction of Clostridium spp. and

Anaerosalibacter from weeks 2 to 14 while uncultured

Tissierella spp. (AB175363_g and EF559164_g) and

Tepidimicrobium was increased at week 6. The relative abundance of the various genera increased at week 6, and this produced the highest diversity of bacteria. After six weeks the leachate might have contained a variety of

or-ganic compounds that supported the growth of diverse classes of bacteria.

The result of the microbial communities grouped by UniFrac and PCoA is shown in Figure 3. The community compositions in the two and four weeks samples were simi-lar, while the compositions changed after six and 14 weeks, which suggests that different bacteria were active at each stage of decomposition. Furthermore, the first principal co-ordinate (PC1, variance of 30.0%) separated the microbial communities in accordance with the pattern of pH suggest-ing that major contributor to affect dynamics of the micro-bial communities might be pH, although other factors af-fecting microbial community did not be shown in this study.

Although most sequences were identified as uncul-tured bacteria at species, the assignment using the Eztaxon-e database provided detailed information about the uncultured bacteria (Figure. 4). Uncultured bacteria

similar toTissierella (Tissierella_f_uc_s) were the most dominant species among all samples, and most of the se-quences identified as unculturedTissierellawere similar to GenBank accession numbers AB175363 and EF559164. UnculturedTissierellaAB175363 is a mesophilic anaero-bic protein-degrading bacterium, and uncultured

TissierellaEF559164 is involved in the methanization of cellulose under mesophilic conditions (Tanget al., 2005; Li

et al., 2009). Therefore, uncultured Tissierella detected here may play a main role in the degradation of pig proteins during 14 weeks. The numbers ofP. russelliiwas increased significantly at week 14, andClostridiumspp. were abun-dant in the week 1 sample.P. russelliiwas isolated from a swine-manure storage pit, and the presence of this species could be related to the decomposition stage of organic ma-terials (Whiteheadet al., 2011).Clostridiumspp. are ubiq-uitous anaerobes found in various environments and are potentially pathogenic.Clostridiumspp. were isolated from the gastrointestinal tract of pigs, and are abundant at rela-tively early stages of decomposition (Varel et al., 1995; Alvarez-Perezet al., 2009; Garciaet al., 2009). Although most of the bacteria detected are anaerobic, the proportion of the aerobic bacterium Comamonas kerstersii was

in-creased at week 6. Species ofComamonasare also present during anaerobic acidification of pig excreta (Zhanget al., 2011). The diverse uncultured bacteria detected in this study provide further information about leachate decompo-sition in the environment.

Recently it was reported that limestone-containing hydrated lime and quicklime affected the decomposition of pig carcasses by changing the pH in burial pits (Schotsmans

et al., 2012). Quicklime was used in the present study to de-compose the pig carcasses, which might have contributed to the sharp increase in pH to 8.79 caused by permeation of the leachate through the layer of quicklime during six weeks (Table 1). This increased pH might be related to the highest level of diversity in this sample. The shifts in bacte-rial communities indicate that different bactebacte-rial species played a role during specific decomposition periods and generated and degraded organic materials that differed dur-ing each period.

Investigations of shifts in the population of bacterial communities are important, because the leachate affects the conditions of adjacent land and the surrounding environ-ment. Thus, microorganism can influence the ecosystem balance through the activities of their metabolites (Stahl

and Tiedje, 2002). Furthermore, the decomposition of car-casses adversely affects the environment for as long as two to ten weeks after burial (McDaniel, 1991). Our present findings indicate a continual shift in the composition of bacterial communities in leachates generated during the de-composition of pig carcasses. The bacterial community in the leachates could affect the surrounding environment, in-cluding soil and groundwater at or near the burial site, po-tentially leading to changes in the composition of organic materials, indigenous microbes, and symbionts. These changes can be expected to adversely affect public health.

In conclusion, we found shifts in bacterial communi-ties in leachates of decomposing pig carcasses during 14 weeks. Different bacterial groups dominated and may play major roles in the decomposition during specific time inter-vals. UnculturedTissiellera spp. and Peptostreptococcus

spp. were present in abundance, and the pH was an interac-tive factor in the decomposition of pig carcasses. The results illustrate the population dynamics of bacterial com-munities and revealed the predominant bacterial species in

such leachates. This information holds promise to improve and implement measures against any future outbreaks of FMD in Korea and other countries and could also be helpful for maintaining the health of ecosystems. Clearly, further studies of burial sites areas will be required to manage and to avert adverse effects on public health.

Acknowledgments

This work was carried out with the support of Coop-erative Research Program for Agriculture Science & Tech-nology Development (Project No. PJ90711004), Rural Development Administration, Republic of Korea. The au-thors wish to thank Mrs. Song Ja Jeong for her help on labo-ratory work.

References

Ahn HK (2012) 2010 FMD outbreak in Korea: Goverment’s re-sponse to this emergency and important lessons learned. Keynote presentation at 4th International Symposium on

managing Animal Mortality, Products, By Products, and As-sociated Health Risk, May 21-24, Dearborn, Michigan, Uni-versity of Maine.

Alvarez-Perez S, Alba P, Blanco JL and Garcia ME (2009) Detec-tion of toxigenic Clostridium difficile in pig feces by PCR. Vet Med-Czech 54:360-366.

Blagodatskaya EV and Anderson TH (1998) Interactive effects of pH and substrate quality on the fungal-to-bacterial ratio and QCO (2) of microbial communities in forest soils. Soil Biol Biochem 30:1269-1274.

Brooksby JB (1982) Portraits of viruses: foot-and-mouth disease virus. Intervirology 18:1-23.

Campobasso CP, Di Vella G and Introna F (2001) Factors affect-ing decomposition and Diptera colonization. Forensic Sci Int 120:18-27.

Carter DO and Tibbett M (2006) Microbial decomposition of skeletal muscle tissue (Ovis aries) in a sandy loam soil at dif-ferent temperatures. Soil Biol Biochem 38:1139-1145. Carter DO, Yellowlees D and Tibbett M (2007) Cadaver

decom-position in terrestrial ecosystems. Naturwissenschaften 94:12-24.

Chen GJ and Russell JB (1989) Sodium-dependent transport of branched-chain smino-acids by a monensin-sensitive ru-minal Peptostreptococcus. Appl Environ Microb 55:2658-2663.

Christensen TH, Kjeldsen P, Albrechtsen HJ, Heron G, Nielsen PH, Bjerg PL and Holm PE (1994) Attenuation of landfill leachate pollutants in aquifers. Crit Rev Env Sci Tec 24:119-202.

Chun J, Kim KY, Lee JH and Choi Y (2010) The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice using a 454 GS FLX Titanium pyrose-quencer. BMC Microbiol 10:e101.

Conant RT, Dalla-Betta P, Klopatek CC and Klopatek JA (2004) Controls on soil respiration in semiarid soils. Soil Biol Biochem 36:945-951.

Domingo E, Mateu MG, Martínez MA, Dopazo J, Moya A and Sobrino F (1990) Genetic variability and antigenic diversity of foot-and-mouth disease virus. In: Kurstak E, Marusyk RG, Murphy SA and VanRegenmortel MHV (eds) Applied Virology Research, vol 2: Virus Variation and Epidemiol-ogy. Plenum Publishing Corp, New York, pp 233-266. Garcia A, Ayuso D, Benitez JM, Garcia WL, Martinez R and

Sanchez S (2009)Clostridium novyiinfection causing sow mortality in an Iberian pig herd raised in an outdoor rearing system in Spain. J Swine Health Prod 17:264-268. Gill-King H (1997) Chemical and ultrastructural aspects of

de-composition. In: Haglund WD and Sorg MH (eds) Forensic Taphonomy: The Postmortem Fate of Human Remains. CRC Press, New York, pp 93-104.

Gruman MJ and Baxt B (2004) Foot-and-mouth disease. Clin Microbiol Rev 17:465-493.

Hamady M, Lozupone C and Knight R (2010) Fast UniFrac: facil-itating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data. ISME J 4:17-27.

Harms C, Schleicher A, Collins MD and Andreesen JR (1998) Tissierella creatinophilasp. nov., a Gram-positive, anaero-bic, non-spore-forming, creatinine-fermenting organism. Int J Syst Bacteriol 48:983-993.

Hur M, Kim Y, Song HR, Kim JM, Choi YI and Yi H (2011) Ef-fect of genetically modified poplars on soil microbial com-munities during the phytoremediation of waste mine tail-ings. Appl Environ Microb 77:7611-7619.

Keener HM, Elwell DL and Monnin MJ (2000) Procedures and equations for sizing of structures and windrows for com-posting animal mortalities. Appl Eng Agric 16:681-692. Kim BS, Kim JN, Yoon SH, Chun J and Cerniglia CE (2012a)

Im-pact of enrofloxacin on the human intestinal microbiota re-vealed by comparative molecular analysis. Anaerobe 18:310-20.

Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S and Chun J (2012b) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence data-base with phylotypes that represent uncultured species. Int J Syst Evol Micr 62:716-721.

Kirchman DL, Cottrell MT and Lovejoy C (2010) The structure of bacterial communities in the western Arctic Ocean as re-vealed by pyrosequencing of 16S rRNA genes. Environ Microbiol 12:1132-1143.

Li TL, Mazeas L, Sghir A, Leblon G and Bouchez T (2009) In-sights into networks of functional microbes catalysing methanization of cellulose under mesophilic conditions. En-viron Microbiol 11:889-904.

Liu ZZ, Lozupone C, Hamady M, Bushman FD and Knight R (2007) Short pyrosequencing reads suffice for accurate mi-crobial community analysis. Nucleic Acids Res 35:e120. Liu ZZ, DeSantis TZ, Andersen GL and Knight R (2008)

Accu-rate taxonomy assignments from 16S rRNA sequences pro-duced by highly parallel pyrosequencers. Nucleic Acids Res 36:e120.

Ludvigsen L, Albrechtsen HJ, Holst H and Christensen TH (1997) Correlating phospholipid fatty acids (PLFA) in a landfill leachate polluted aquifer with biogeochemical factors by multivariate statistical methods. FEMS Microbiol Rev 20:447-460.

Lynch JM and Hobbie JE (1988) Micro-Organisms in Action: Concepts and Applications in Microbial Ecology. Blackwell Scientific Publications, Oxford, 363 pp.

Mann RW, Bass WM and Meadows L (1990) Time since death and decomposition of the human body: variables and obser-vations in case and experimental field studies. J Forensic Sci 35:103-111.

Matthies C, Erhard HP and Drake HL (1997) Effects of pH on the comparative culturability of fungi and bacteria from acidic and less acidic forest soils. J Basic Microb 37:335-343. McDaniel HA (1991) Environmental protection during animal

disease eradication programmes. Rev Sci Tech OIE 10:867-884.

NRC (2002) Biosolids Applied to Land: Advancing Standards and Practices. National Research Council, Washington DC, 344 pp.

Paul EA and Clark FE (1996) Soil Microbiology and Biochemis-try. 2nd edition. Academic Press, San Diego, 340 pp. Pfeiffer S, Milne S and Stevenson RM (1998) The natural

decom-position of adipocere. J Forensic Sci 43:368-370.

Rezgui R, Maaroufi A, Fardeau ML, Ben Ali Gam Z, Cayol JL, Ben Hamed S and Labat M (2012) Anaerosalibacter bizertensisgen. nov., sp. nov., a new halotolerant bacterium isolated from sludge. Int J Syst Evol Micr 60:2469-2474. Röling WFM, van Breukelen BM, Braster M, Lin B and van

Verseveld HW (2001) Relationships between microbial community structure and hydrochemistry in a landfill leach-ate-polluted aquifer. Appl Environ Microb 67:4619-4629. Sasaki H, Nonaka J, Otawa K, Kitazume O, Asano R, Sasaki T

and Nakai Y (2009) Analysis of the structure of the bacterial community in the livestock manure-based composting pro-cess. Asian-Aust J Anim 22:113-118.

Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hol-lister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, et al. (2009) Introducing mothur: open-source, plat-form-independent, community-supported software for de-scribing and comparing microbial communities. Appl Envi-ron Microb 75:7537-7541.

Schotsmans EM, Denton J, Dekeirsschieter J, Ivaneanu T, Leentjes S, Janaway RC and Wilson AS (2012) Effects of hydrated lime and quicklime on the decay of buried human remains using pig cadavers as human body analogues. Fo-rensic Sci Int 217:50-9.

Seellers RF (1995) Growth and titration of the viruses of foot-and mouth disease and vesicular stomatitis in kidney monolayer tissue cultures. Nature 176:121-124.

Sharma PK and Das SK (1984) Occurrence of foot-and mouth dis-ease and distribution of virus type in the hill states of North Eastern region of India. Indian J Anim Sci 4:117-118. Singleton DR, Furlong MA, Rathbun SL and Whitman WB

(2001) Quantitative comparisons of 16S rRNA gene se-quence libraries from environmental samples. Appl Environ Microb 67:4374-4376.

Slobodkin AI, Tourova TP, Kostrikina NA, Lysenko AM, Ger-man KE, Bonch-Osmolovskaya EA and Birkeland NK (2006) Tepidimicrobium ferriphilum gen nov, sp nov, a novel moderately thermophilic, Fe(III)-reducing bacterium of the order Clostridiales. Int J Syst Evol Micr 56:369-372. Stahl DA and Tiedje JM (2002) Microbial Ecology and

Geno-mics: A Crossroads of Opportunity. Am Acad Microbiol, Washington. DC, 28 pp.

Tang YQ, Shigematsu T, Morimura S and Kida K (2005) Micro-bial community analysis of mesophilic anaerobic protein degradation process using bovine serum albumin (BSA)-fed continuous cultivation. J Biosci Bioeng 99:150-164. Tian Y, Yang H, Wu X and Li DT (2005) Molecular analysis of

microbial community in a groundwater sample polluted by landfill leachate and seawater. J Zhejiang Univ Sci 6:165-170.

Van’tHoff JH (1898) Lectures on Theoretical and Physical Chem-istry. Part 1. Chemical Dynamics. Edward Arnold, London, pp. 224-229.

Varel VH, Tanner RS and Woese CR (1995) Clostridium herbivoranssp nov, a cellulolytic anaerobe from the pig in-testine. Int J Syst Bacteriol 45:490-494.

Vass AA, Bass WM, Wolt JD, Foss JE and Ammons JT (1992) Time since death determinations of human cadavers using soil solution. J Forensic Sci 37:1236-1253.

Whitehead TR, Cotta MA, Falsen E, Moore E and Lawson PA (2011)Peptostreptococcus russelliisp nov., isolated from a swine-manure storage pit. Int J Syst Evol Micr 61:1875-1879.

Woodbury EL (1995) A review of the possible mechanisms for the persistence of foot-and-mouth disease virus. Epidemiol Infect 114:1-13.

Yang SH, Hong SH, Cho SB, Lim JS, Bae SE, Ahn HK and Lee EY (2012) Characterization of microbial community in the leachate associated with the decomposition of entombed pigs. J Microbiol Biotechn 22:1330-5.

Zhang DD, Yuan XF, Guo P, Suo YL, Wang XF, Wang WD and Cui ZJ (2011) Microbial population dynamics and changes in main nutrients during the acidification process of pig ma-nures. J Environ Sci (China) 23:497-505.

Internet Resources

EzTaxon-e database, http://eztaxon-e.ezbiocloud.net (accessed at 2012.10.02).

Associate Editor: Carlos F.M. Menck